Injectable bladder muscle cells-polymer suspension for treatment of vesicoureteral reflux and incontinence

ABSTRACT

A method of treatment of vesicoureteral reflux, incontinence and other defects is described wherein bladder muscle cells are mixed with a liquid polymeric material to form a suspension. This is injected into the area of the defect, in an amount effective to yield a muscle area that provides the required control over the passage of urine or otherwise corrects the defect.

This is a continuation-in-part of U.S. Ser. No. 08/056,140 filed Apr.30, 1993 by Keith T. Paige, Linda G. Cima, Charles A. Vacanti andAnthony Atala entitled "Injectable Polysaccharide-Cell Compositions".

BACKGROUND OF THE INVENTION

The present invention is generally in the area of medical treatments,and specifically relates to a method for correcting vesicoureteralreflux, incontinence and other defects.

Vesicoureteral reflux.

Vesicoureteral reflux is a condition wherein there is an abnormaldevelopment of the ureteral bud as it enters the bladder duringembryologic development. The shortened course of the ureter through thebladder musculature decreases the ureteral resistance and allows forurine to reflux from the bladder reservoir back up into the ureter andinto the kidney. With this condition, bacteria which may occasionally bepresent in the bladder through retrograde urethral transport, can reachthe kidneys and cause recurrent pyelonephritis. In addition, theconstant back pressure of the urine into the calyces and renal pyramidsresults in mechanical damage to the renal parenchyma. If untreated,urinary vesicoureteral reflux can cause loss of renal parenchyma, and insome instances, renal failure, as reviewed by Atala and Casale,Infections in Urology 39-43 (March/April 1990). In 1960, 70% of thepatients with renal failure were described as having vesicoureteralreflux as the primary etiology. With the advent of new diagnostic andtreatment modalities, patients with vesicoureteral reflux now accountfor less than 1% of the renal failure population.

In the past, vesicoureteral reflux was usually diagnosed with a voidingcystogram after the child presented with repeated episodes ofpyelonephritis. With the increased use of prenatal and postnatalsonography, hydronephrosis is more detectable, prompting furtherradiologic workup and earlier detection, as reported by Atala andCasale. Vesicoureteral reflux is graded depending on the severity. Grade1 reflux signifies that urine is seen refluxing from the bladder up tothe ureter only; in grade 2 reflux, urine refluxes into the ureter andcalyceal dilatation. Grade 4 and 5 reflux are more severe, showingureteral tortuosity and further calyceal blunting and dilatation,respectively.

The treatment of vesicioureteral reflux has been well established overthe last decade. Initially it was believed that all patients with refluxwould require surgery. Another school of management soon proposed thatonly medical therapy with antibiotics was required. It is now wellestablished that the treatment of reflux depends on many factors,including the severity of reflux, associated congenital abnormalities,and the social situation of the child (parental compliance with medicaltreatment). Medical treatment is usually recommended for patients withgrade 1 and 2 reflux, which usually resolve on their own as thebladder/ureteral configuration changes with growth. Grade 3 reflux isgenerally managed with medical therapy unless it persists orbreakthrough infections occur while on antibiotic suppression. Surgicaltreatment is usually required for grade 4 and 5 reflux.

Medical treatment implies that the patient is treated with dailysuppressive antibiotics. A close follow-up is required in thesepatients, generally consisting of a catheterized urine culture everythree months, an ultrasound exam and serum analysis every six months, afluoroscopic or nuclear voiding cystourethrogram every year, and a DMSArenal scan every two years. Surgical treatment consists of an opensurgery wherein a low abdominal incision is made, the bladder isentered, the ureters are mobilized and new ureteral submucosal tunnelsare created; thereby extending the muscular backing of the ureter whichincreases their resistance. These patients require a generalendotracheal anesthetic for a four to five hour surgery, an epiduralcatheter for both intraoperative and postoperative pain control, abladder catheter for drainage, a perivesical drain, and a five to sixday hospital stay. Antibiotic therapy and bladder antispasmodics arerequired post-operatively.

Although open surgical procedures for the correction of reflux haveexcellent results in the hands of experienced surgeons, it is associatedwith a well recognized morbidity, including pain and immobilization of alower abdominal incision, bladder spasms, hematuria, and post-operativevoiding frequency in some children. In an effort to avoid open surgicalintervention, widespread interest was initiated by Matouschek's clinicalexperience with the endoscopic injection of Teflon™(polytetrafluoroethylene) paste subureterally in 1984, as reported inMatouschek, E.: Die Behandlung des vesikorenalen Refluxes durchtransueterale Einspritzung von polytetrafluoroethylenepast. Urologe,20:263 (1981). With this technique, a cystoscope is inserted into thebladders, a needle is inserted through the cystoscope and placed underdirect vision underneath the refluxing ureter in the submucosal space,and Teflon™ paste is injected until the gaping ureteric orificeconfiguration changes into a half-moon slit. The Teflon™ paste, injectedendoscopically, corrects the reflux by acting as a bulking materialwhich increases ureteral resistance. However, soon after theintroduction of this treatment, a controversy regarding the use ofTeflon™ paste ensued. Malizia et al. "Migration and granulomatousreaction after periurethral injection of polymer(polytetrafluoroethylene)" JAMA, 251:3277 (1984), showed granulomaformation and particulate migration to the brain, lungs, and lymph nodesin animal studies. Polytetrafluoroethylene migration and granulomaformation have also been reported in humans by Claes et al., "Pulmonarymigration following periurethral polyetrafluoroethylene injection forurinary incontinence" J. Urol., 142:821 (1989). The safety of Teflon™for human use was questioned, and the paste was thereafter banned by theFDA.

However, there are definite advantages in treating vesicoureteral refluxendoscopically. The method is simple and can be completed in less thanfifteen minutes, it has a success rate of greater than 85% with lowmorbidity and it can be performed in an outpatient basis, as reported byAtala et al, "Endoscopic treatment of vesicoureteral reflux with aself-detachable balloon system" J. Urol. 148:724 (1992). The goal ofseveral investigators has been to find alternate implant materials whichwould be safe for human use.

Bovine dermal collagen preparations have been used to treat refluxendoscopically. However, only 58.5% of the patients were cured at oneyear follow-up, as described by Leonard et al, "Endoscopic injection ofglutaraldehyde cross-linked bovine dermal collagen for correction ofvesicoureteral reflux" J. Urol. 145:115 (1991). The collagen implantvolume decreases with time, which results in high percentage ofrecurrence of reflux, over 90% within 3 years. The high failure ratewith this substance presents a high risk to the unaware patient ofdeveloping renal damage after treatment.

A paste consisting of textured microparticles of silicone, suspended ina hydrogel, has been injected subureterally to correct reflux with aninitial success rate of 91% in one European study, as reported byBuckley et al., "Endoscopic correction of vesicoureteric reflux withinjectable silicone microparticles" J. Urol. 149:259A (1993). However,distant particle migration has been observed in animal models, asreported by Henly et al., "Particulate silicone for use in periurethralinjections: a study of local tissue effects and a search for migration"J. Urol. 147:376A (1992). Approximately thirty percent of the siliconeparticles have a diameter which is less than 100 μm. This suggests thatthirty percent of the silicone particles have a potential for distantorgan migration through the macrophage system. The manufacturer of thistechnology tried unsuccessfully to obtain FDA approval, and subsequentlyfiled for bankruptcy.

Laparoscopic correction of reflux has been attempted in both an animalmodel (Atala et al, "Laparoscopic correction of vesicoureteral reflux"J. Urol. 150:748 (1993)) and humans (Atala, "Laparoscopic treatment ofvesicoureteral reflux"Dial Ped Urol 14:212 (1993)) and is technicallyfeasible. However, at least two surgeons with laparoscopic expertise areneeded, the length of the procedure is much longer than with opensurgery, the surgery is converted from an extraperitoneal to anintraperitoneal approach, and the cost is higher due to both increasedoperative time and the expense of the disposable laparoscopic equipment.

Despite the fact that over a decade has transpired since the Teflon™controversy, little progress has been made in this area of research. Theideal substance for the endoscopic treatment of reflux should beinjectable, non-antigenic, non-migratory, volume stable, and safe forhuman use (Atala et al, 1992).

Urinary incontinence.

Urinary Incontinence is the most common and the most intractable of allGU maladies. Urinary incontinence, or the inability to retain urine andnot void urine involuntarily, is dependent on the interaction of twosets of muscles. One is the detrusor muscle, a complex of longitudinalfibers forming the external muscular coating of the bladder. Thedetrusor is activated by parasympathetic nerves. The second muscle isthe smooth/striated muscle of the bladder sphincter. The act of voidingrequires the sphincter muscle be voluntarily relaxed at the same timethat the detrusor muscle of the bladder contracts. As a person ages, hisability to voluntarily control the sphincter muscle is lost in the sameway that general muscle tone deteriorates with age. This can also occurwhen a radical event such as paraplegia "disconnects" theparasympathetic nervous system causing a loss of sphincter control. Indifferent patients, urinary incontinence exhibits different levels ofseverity and is classified accordingly.

The most common incontinence, particular in the elderly, is urgeincontinence. This type of incontinence is characterized by an extremelybrief warning following by immediate urination. This type ofincontinence is caused by a hyperactive detrusor and is usually treatedwith "toilet training" or medication. Reflex incontinence, on the otherhand, exhibits no warning and is usually the result of an impairment ofthe parasympathetic nerve system such as a spinal cord injury.

Stress incontinence is most common in elderly women but can be found inwomen of any age. It is also commonly seen in pregnant women. This typeof incontinence accounts for over half of the total number of cases. Itis also found in men but at a lower incidence. Stress incontinence ischaracterized by urine leaking under conditions of stress such assneezing, laughing or physical effort. There are five recognizedcategories of severity of stress incontinence, designated as types as 0,1, 2a, 2b, and 3. Type 3 is the most severe and requires a diagnosis ofintrinsic Sphincter Deficiency or ISD (Contemporary Urology, March1993). There are many popular treatments including weight loss,exercise, medication and in more extreme cases, surgical intervention.The two most common surgical procedures involve either elevating thebladder neck to counteract leakage or constructing a lining from thepatient's own body tissue or a prosthetic material such as PTFE to putpressure on the urethra. Another option is to use prosthetic devicessuch as artificial sphincters to external devices such as intravaginalballoons or penile clamps. For treatment of type 3 stress incontinence,there has been a recent trend toward injection of Teflon™ or collagenpaste around the sphincter muscle in order to "beef up" the area andimprove muscle tone. None of the above methods of treatment, however,are very effective for periods in excess of a year.

Overflow incontinence is caused by anatomical obstructions in thebladder or underactive detrustors. It is characterized by a distendedbladder which leads to frequent urine leakage. This type of incontinenceis treated acutely by catheterization and long-term by drug therapy.Enuresis or bed-wetting is a problem in pediatrics and is controlled byvarious alarming devices and pads with sensors. Enuresis is notconsidered a serious problem unless it lasts beyond the age of four orfive. Finally, there is true functional incontinence which occurs inpatients with chronic impairment either of mobility or mental function.Such patients are usually treated by the use of diapers, incontinencepads or continuous catheterization (BBI, 1985 Report 7062).

It is therefore an object of the present invention to provide a methodand material for treating vesicoureteral reflux, incontinence, and otherdefects which results in a natural and permanent cure to the defect.

It is a further object of the present invention to provide a method andmaterial for treating vesicoureteral reflux, incontinence, and otherdefects which is quick, simple, safe, and relatively non-invasive.

SUMMARY OF THE INVENTION

A method of treatment of vesicoureteral reflux, incontinence and otherdefects is described wherein bladder muscle cells are mixed with aliquid polymeric material, such as alginate which can be solidified invivo, to form a cell suspension, which is injected into the area of thedefect, in an amount effective to yield a muscle area that corrects thedefect, for example, which provides the required control over thepassage of urine.

As described in the examples, human bladder muscle specimens areobtained and processed within one hour after surgical removal. In thepreferred embodiment, muscle cells are mixed with a biodegradable liquidpolymer such as alginate, a biodegradable copolymer of gluronic andmannuronic acid, which is designed to solidify at a controlled rate whencontacted with calcium salts. The cells are then injected at the desiredsite where they proliferate and correct the defect. Examples demonstrateefficacy in mice.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic of injection of a polymer-muscle cell suspensioninto a region for control of vesicoureteral reflux.

DETAILED DESCRIPTION OF THE INVENTION

A biodegradable polymer, embedded with muscle cells, serves as asynthetic substrate for the injectable delivery and maintenance ofmuscle architecture in humans that satisfies all the requirements for anideal injectable substance. A biopsy of the bladder can be easily andquickly performed cystoscopically followed by muscle cell processing andendoscopic injection of the autologous muscle cell/polymer suspensionfor the treatment reflux, incontinence and other defects.

Studies show that muscle cells can be easily harvested and combined withalginate in vitro, the suspension can be easily injected cystoscopicallyand the muscle tissue formed is able to correct vesicoureteral refluxwithout any evidence of obstruction. The ideal injectable substance forthe endoscopic treatment of reflux should be a natural bulking agentwhich is non-antigenic, non-migratory, and volume stable. Autologousmuscle cells seem to fulfill all of these requirements. Since the musclecells are autologous, this method of treatment does not require FDAapproval. The procedure can be performed under 15 minutes, with a shortperiod of a mask anesthetic, in the outpatient unit, without any needfor a hospital stay. Neither vesical nor perivesical drainage isrequired. Since the whole procedure is done endoscopically and thebladder is not entered surgically, there is no postoperative discomfortwhatsoever. The patient can return to a normal level of activity almostimmediately.

Source of Cells

In the preferred embodiment, cells of the same species and preferablyimmunological profile are obtained by biopsy, either from the patient ora close relative, which are then grown to confluence in culture usingstandard conditions as described below in Example 1 and used as needed.If cells that are likely to elicit an immune reaction are used, such ashuman muscle cells from immunologically distinct individual, then therecipient can be immunosuppressed as needed, for example, using aschedule of steroids and other immunosuppressant drugs such ascyclosporine. However, in the most preferred embodiment, the cells areautologous.

Cells obtained by biopsy are harvested and cultured, passaging asnecessary to remove contaminating non-bladder muscle cells.

Polymer Solutions

In the preferred embodiment described herein, calcium alginate andcertain other polymers that can form ionic hydrogels which are malleableare used to encapsulate cells. The hydrogel is produced by cross-linkingthe anionic salt of alginic acid, a carbohydrate polymer isolated fromseaweed, with calcium cations, whose strength increases with eitherincreasing concentrations of calcium ions or alginate. The alginatesolution is mixed with the cells to be implanted to form an alginatesuspension. Then the suspension is injected directly into a patientprior to hardening of the suspension. The suspension then hardens over ashort period of time due to the presence in vivo of physiologicalconcentrations of calcium ions.

The polymeric material which is mixed with cells for implantation intothe body should form a hydrogel. A hydrogel is defined as a substanceformed when an organic polymer (natural or synthetic) is cross-linkedvia covalent, ionic, or hydrogen bonds to create a three-dimensionalopen-lattice structure which entraps water molecules to form a gel.Examples of materials which can be used to form a hydrogel includepolysaccharides such as alginate, polyphosphazines, and polyacrylates,which are crosslinked ionically, or block copolymers such as Pluronics™or Tetronics™, polyethylene oxide-polypropylene glycol block copolymerswhich are crosslinked by temperature or pH, respectively. Othermaterials include proteins such as fibrin, polymers such aspolyvinylpyrrolidone, hyaluronic acid and collagen.

In general, these polymers are at least partially soluble in aqueoussolutions, such as water, buffered salt solutions, or aqueous alcoholsolutions, that have charged side groups, or a monovalent ionic saltthereof. Examples of polymers with acidic side groups that can bereacted with cations are poly(phosphazenes), poly(acrylic acids),poly(methacrylic acids), copolymers of acrylic acid and methacrylicacid, poly(vinyl acetate), and sulfonated polymers, such as sulfonatedpolystyrene. Copolymers having acidic side groups formed by reaction ofacrylic or methacrylic acid and vinyl ether monomers or polymers canalso be used. Examples of acidic groups are carboxylic acid groups,sulfonic acid groups, halogenated (preferably fluorinated) alcoholgroups, phenolic OH groups, and acidic OH groups.

Examples of polymers with basic side groups that can be reacted withanions are poly(vinyl amines), poly(vinyl pyridine), poly(vinylimidazole), and some imino substituted polyphosphazenes. The ammonium orquaternary salt of the polymers can also be formed from the backbonenitrogens or pendant imino groups. Examples of basic side groups areamino and imino groups.

Alginate can be ionically cross-linked with divalent cations, in water,at room temperature, to form a hydrogel matrix. Due to these mildconditions, alginate has been the most commonly used polymer forhybridoma cell encapsulation, as described, for example, in U.S. Pat.No. 4,352,883 to Lim. In the Lim process, an aqueous solution containingthe biological materials to be encapsulated is suspended in a solutionof a water soluble polymer, the suspension is formed into droplets whichare configured into discrete microcapsules by contact with multivalentcations, then the surface of the microcapsules is crosslinked withpolyamino acids to form a semipermeable membrane around the encapsulatedmaterials.

Polyphosphazenes are polymers with backbones consisting of nitrogen andphosphorous separated by alternating single and double bonds. Eachphosphorous atom is covalently bonded to two side chains ("R"). Therepeat unit in polyphosphazenes has the general structure (1): ##STR1##

The polyphosphazenes suitable for cross-linking have a majority of sidechain groups which are acidic and capable of forming salt bridges withdi- or trivalent cations. Examples of preferred acidic side groups arecarboxylic acid groups and sulfonic acid groups. Hydrolytically stablepolyphosphazenes are formed of monomers having carboxylic acid sidegroups that are crosslinked by divalent or trivalent cations such asCa²⁺ or Al³⁺. Polymers can be synthesized that degrade by hydrolysis byincorporating monomers having imidazole, amino acid ester, or glycerolside groups. For example, a polyanionicpoly[bis(carboxylatophenoxy)]phosphazene (PCPP) can be synthesized,which is cross-linked with dissolved multivalent cations in aqueousmedia at room temperature or below to form hydrogel matrices.

Bioerodible polyphosphazines have at least two differing types of sidechains, acidic side groups capable of forming salt bridges withmultivalent cations, and side groups that hydrolyze under in vivoconditions, e.g., imidazole groups, amino acid esters, glycerol andglucosyl. The term bioerodible or biodegrable, as used herein, means apolymer that dissolves or degrades within a period that is acceptable inthe desired application (usually in vivo therapy), less than about fiveyears and most preferably less than about one year, once exposed to aphysiological solution of pH 6-8 having a temperature of between about25° C. and 38° C. Hydrolysis of the side chain results in erosion of thepolymer. Examples of hydrolyzing side chains are unsubstituted andsubstituted imidizoles and amino acid esters in which the group isbonded to the phosphorous atom through an amino linkage (polyphosphazenepolymers in which both R groups are attached in this manner are known aspolyaminophosphazenes). For polyimidazolephosphazenes, some of the "R"groups on the polyphosphazene backbone are imidazole rings, attached tophosphorous in the backbone through a ring nitrogen atom. Other "R"groups can be organic residues that do not participate in hydrolysis,such as methyl phenoxy groups or other groups shown in the scientificpaper of Allcock, et al., Macromolecule 10:824-830 (1977).

Methods for synthesis and the analysis of various types ofpolyphosphazenes are described by Allcock, H. R.; et al., Inorg. Chem.11, 2584 (1972); Allcock, et al., Macromolecules 16, 715 (1983);Allcock, et al., Macromolecules 19, 1508 (1986); Allcock, et al.,Biomaterials, 19, 500 (1988); Allcock, et al., Macromolecules 21, 1980(1988); Allcock, et al., Inorg. Chem. 21(2), 515-521 (1982); Allcock, etal., Macromolecules 22, 75 (1989); U.S. Pat. Nos. 4,440,921, 4,495,174and 4,880,622 to Allcock, et al.; U.S. Pat. No. 4,946,938 to Magill, etal.; and Grolleman, et al., J. Controlled Release 3, 143 (1986), theteachings of which are specifically incorporated herein by reference.

Methods for the synthesis of the other polymers described above areknown to those skilled in the art. See, for example Concise Encyclopediaof Polymer Science and Polymeric Amines and Ammonium salts, E. Goethals,editor (Pergamen Press, Elmsford, N.Y. 1980). Many polymers, such aspoly(acrylic acid), are commercially available.

The water soluble polymer with charged side groups is crosslinked byreacting the polymer with an aqueous solution containing multivalentions of the opposite charge, either multivalent cations if the polymerhas acidic side groups or multivalent anions if the polymer has basicside groups. The preferred cations for cross-linking of the polymerswith acidic side groups to form a hydrogel are divalent and trivalentcations such as copper, calcium, aluminum, magnesium, strontium, barium,and tin, although di-, tri- or tetra-functional organic cations such asalkylammonium salts, e.g., R₃ N⁺ --\/\/\/--⁺ NR₃ can also be used.Aqueous solutions of the salts of these cations are added to thepolymers to form soft, highly swollen hydrogels and membranes. Thehigher the concentration of cation, or the higher the valence, thegreater the degree of cross-linking of the polymer. Concentrations fromas low as 0.005M have been demonstrated to cross-link the polymer.Higher concentrations are limited by the solubility of the salt.

The preferred anions for cross-linking of the polymers to form ahydrogel are divalent and trivalent anions such as low molecular weightdicarboxylic acids, for example, terepthalic acid, sulfate ions andcarbonate ions. Aqueous solutions of the salts of these anions are addedto the polymers to form soft, highly swollen hydrogels and membranes, asdescribed with respect to cations.

A variety of polycations can be used to complex and thereby stabilizethe polymer hydrogel into a semi-permeable surface membrane. Examples ofmaterials that can be used include polymers having basic reactive groupssuch as amine or imine groups, having a preferred molecular weightbetween 3,000 and 100,000, such as polyethylenimine and polylysine.These are commercially available. One polycation is poly(L-lysine);examples of synthetic polyamines are: polyethyleneimine,poly(vinylamine), and poly(allyl amine). There are also naturalpolycations such as the polysaccharide, chitosan.

Polyanions that can be used to form a semi-permeable membrane byreaction with basic surface groups on the polymer hydrogel includepolymers and copolymers of acrylic acid, methacrylic acid, and otherderivatives of acrylic acid, polymers with pendant SO₃ H groups such assulfonated polystyrene, and polystyrene with carboxylic acid groups.

Cell Suspensions

Preferably the polymer is dissolved in an aqueous solution, preferably a0.1M potassium phosphate solution, at physiological pH, to aconcentration forming a polymeric hydrogel, for example, for alginate,of between 0.5 to 2% by weight, preferably 1%, alginate. The isolatedbladder muscle cells are suspended in the polymer solution to aconcentration of between 1 and 50 million cells/ml, most preferablybetween 10 and 20 million cells/ml.

Injection of Cells

Vesicoureteral reflux is one of the most common congenital defects inchildren, affecting approximately 1% of the population. Although allpatients do not require surgical treatment, it is still one of the mostcommon procedure performed in children. Over 600 ureteral reimplants areperformed yearly at Children's Hospital in Boston, Mass. This translatesto an approximately saving of 3600 inpatient hospital days per year atthis institution alone, if the endoscopic treatment described herein isused instead of open surgery.

In addition to its use for the endoscopic treatment of reflux, thesystem of injectable autologous muscle cell may also be applicable forthe treatment of other medical conditions, such as urinary and rectalincontinence, dysphonia, plastic reconstruction, and wherever aninjectable permanent biocompatible material is needed.

As described herein, an injectable biodegradable polymer as a deliveryvehicle for muscle cells is useful in the treatment of reflux andincontinence. In the preferred embodiment, a bladder muscle biopsy isobtained under anesthesia from a patient with vesicoureteral reflux, theisolated muscle cells are mixed with alginate, and the musclecell-alginate solution is injected endoscopically in the sub-ureteralregion to correct reflux, as shown in FIG. 1. The time to solidificationof the alginate-cell solution may be manipulated by varying theconcentration of calcium as well as the temperature at which the musclecells are added to the alginate. The use of autologous bladder musclecells cells precludes an immunologic reaction. Solidification of thealginate impedes its migration until after it is degraded. Thesuspension can be injected through a cystoscopic needle, having directvisual access with a cystoscope to the area of interest, such as for thetreatment of vesicoureteral reflux or urinary incontinence. In additionto the use of the muscle cell-polymer suspension for the treatment ofreflux and incontinence, the suspension can also be applied toreconstructive surgery, as well as its application anywhere in the humanbody where a biocompatible permanent injectable material is necessary.The suspension can be injected endoscopically, for example through alaryngoscope for injection into the vocal chords for the treatment ofdysphonia, or through a hysteroscope for injection into the fallopiantubes as a method of rendering the patient infertile, or through aproctoscope, for injection of the substance in the perirectal sphincterarea, thereby increasing the resistance in the sphincter area andrendering the patient continent of stool.

The suspension can be injected via a syringe and needle directly into aspecific area wherever a bulking agent is desired, i.e., a soft tissuedeformity such as that seen with areas of muscle atrophy due tocongenital or acquired diseases or secondary to trauma, burns, and thelike. An example of this would be the injection of the suspension in theupper torso of a patient with muscular atrophy secondary to nervedamage.

The suspension can also be injected as a bulking agent for hard tissuedefects, such as bone or cartilage defects, either congenital oracquired disease states, or secondary to trauma, burns, or the like. Anexample of this would be an injection into the area surrounding theskull where a bony deformity exists secondary to trauma. The injunctionin these instances can be made directly into the needed area with theuse of a needle and syringe under local or general anesthesia.

The suspension could also be injected percutaneously by directpalpation, such as by placing a needle inside the vas deferens andoccluding the same with the injected bulking substance, thus renderingthe patient infertile. The suspension could also be injected through acatheter or needle with fluoroscopic, sonographic, computed tomography,magnetic resonance imaging or other type of radiologic guidance. Thiswould allow for placement or injection of this substance either byvascular access or percutaneous access to specific organs or othertissue regions in the body, wherever a bulking agent would be required.

Further, this substance could be injected through a laparoscopic orthoracoscope to any intraperitoneal or extraperitoneal or thoracicorgan. For example, the suspension could be injected in the region ofthe gastro-esophageal junction for the correcting of gastroesophagealreflux. This could be performed either with a thoracoscope injecting thesubstance in the esophageal portion of the gastroesophageal region, orvia a laparoscope by injecting the substance in the gastric portion ofthe gastroesophageal region, or by a combined approach.

The present invention will be further understood by reference to thefollowing non-limiting examples. The examples demonstrate that humanbladder muscle cell-alginate suspensions are injectable, non-migratory,and appear to conserve their volume and that autologous bladder musclecells are useful in the endoscopic treatment of vesicoureteral reflux.

EXAMPLE 1

Isolation and Characterization of Human Bladder Muscle Cells Expanded InVitro.

Cell Culture:

Human Tissue Origin. Human bladder tissue specimens were obtained andprocessed within one hour after surgical removal at Children's Hospital,Boston. The specimens varied in size, ranging from one cm² to four cm².The specimens were transported to Hanks balanced salt solutioncontaining 10 mM HEPES and 100 KIU apoprotein.

Smooth Muscle Cells. Muscle cells were obtained by mincing smalldetrusor muscle fragments to approximately 0.5 mm diameter and usingthese as explants in 100 mm tissue culture dishes containing 10 mL ofDMEM supplemented with 10% fetal calf serum. Approximately 30 explantsof similar size were added to each dish. Medium was changed twice aweek. Outgrowth was routinely observed at 72 hr after explants wereplaced in culture. When cultures were 80% confluent, the cells weretrypsinized and passaged. Cell populations highly enriched in elongated,striated cells were routinely obtained using this method. Cell strainswere stained with a-actin antibody to verify their muscle phenotype.

Results. Populations of fusiform, striated cells could be obtained fromthe bladder biopsy specimens by dissection of the muscle layer followedby explant culture of the muscle fragments in DMEM supplemented with 10%calf-serum. Immunostaining with a monoclonal antibody which specificallyrecognized a-actin verified the muscle phenotype. These data indicatethat highly proliferative populations of smooth muscle can be obtainedfrom small biopsy specimens. The muscle cells can also be growntransiently in serum-free defined media which renders the cells freefrom any impurities.

EXAMPLE 2

Injection of cell suspensions into mice.

Using a 22 gauge needle, 20 nude mice were injected with a 500microliter solution of muscle cells and alginate. Each mouse had twoinjection sites consisting of either control, or a solution of tenmillion human bladder cells per cc of alginate (32 injection sites).Injections of alginate alone or bladder muscle cells alone served ascontrols. Animals were sacrificed at two, four, six and eight weeksafter implantation. Histologic examination of the injection areasdemonstrated evidence of muscle formation in the muscle/alginateinjection sites. Immunohistochemical analysis using an anti-desminantibody indicated that the cells maintained a program of musculardifferentiation. Examination of the injection sites with increasingperiods of time, showed that the alginate was progressively replaced bymuscle. The size of the muscle-alginate complex appeared to be uniformand stable. In both control groups (alginate alone or muscle cellsalone) there were no muscle cells evident. Histologic analysis ofdistant organs showed no evidence of bladder muscle cells or alginatemigration or granuloma formation.

EXAMPLE 3

Correction of vesicouretral reflux in pigs using bladder muscle cellsimplanted in an alginate gel.

Materials and Methods

Animal model of vesicoureteral reflux. The pig was used for this studybecause of the similarities between porcine and human bladders andkidneys. The Hanford mini-pig was used for the convenience of itssmaller size. Bilateral vesicoureteral reflux was created in fourmini-swine using the open bladder technique, which consists of unroofingthe entire intravesical ureter, as described by Vacanti, et al.,"Synthetic polymers seeded with chondrocytes provide a template for newcartilage formation" Plastic and Recon. Surg. 88:753 (1991).

Three months after the procedure, the presence of bilateral reflux wasassessed by conventional radiographic cystography using an iodinatedcontrast agent, and by sonography using sonicated albumin, as describedby Vacanti, et al., "Tissue engineered growth of new cartilage in theshape of a human ear using synthetic polymers seeded with chondrocytes"Mat. Res. Soc. Proc. 252:367 (1992). Excretory urography was performedto detect any evidence of obstruction.

Cell Harvest. A segment of bladder muscle was obtained from each animal.Muscle cells were harvested and plated separately in vitro. Afterexpansion, the cells were individually quantitated and concentrated to20×10⁶ cells per cc.

Autologous bladder muscle cell-calcium alginate suspension. Two percentweight/volume sodium alginate (0.1M K₂ PO₄, 0.135M NaCl, pH 7.4, Protan,Portsmouth, N.H.) was made and sterilized in ethylene oxide. A 1.5 mlaliquot of 20×10⁶ cells/ml bladder muscle cell suspension was added toan equal volume of sodium alginate solution for a final alginateconcentration of 1%. The bladder muscle cell-sodium alginate suspensionwas kept at 32° C. Immediately prior to injection, calcium sulfate (0.2g/ml) was added to the bladder muscle cell-sodium alginate suspension.The mixture was vortexed and stored in ice until injection. The gellingprocess was initiated with the addition of calcium sulfate, whichallowed the suspension to remain in a liquid state for approximately 40minutes.

Experimental study. Mini-pigs were anesthetized with intramuscularinjections of 25 ml/kg ketamine and 1 ml/kg acylpromazine. Additionalanesthesia was obtained with an intramuscular administration of 25 mg/kgketamine and 10 mg/kg of xylazine. Animals were placed in a supineposition. With a 15.5 French cystoscope introduced into the bladder, a21 gauge needle was inserted in the subureteral region of the rightrefluxing ureter. Approximately 2 to 3 ml of the autologous bladdermuscle cell-alginate suspension (40-60×10⁶ bladder muscle cells) wereinjected through the needle, while lifting of the ureteral orifice wasendoscopically visualized. The left ureteral orifice remained untreatedand served as a control. Serial cystograms, cystoscopy, and excretoryurographic studies were performed at eight week intervals untilsacrifice. The mini-pigs were sacrificed at eight (1), 16 (1), and 26(2) weeks after treatment. The bladder injection sites were resected andexamined macroscopically and microscopically. Specimens were stainedwith hematoxylin and eosin, and alcian blue at a pH of 1.0 and 2.5.Histological analyses of the bladder, ureters, regional lymph nodes,kidneys, liver, and spleen were performed.

Results

Four mini-swine underwent bilateral creation of reflux. All four werefound to have bilateral reflux without evidence of obstruction at threemonths following the procedure. Bladder muscle cells were from eachmini-swine and expanded in vitro. The animals then underwent endoscopicrepair of reflux with the injectable autologous bladder musclecell-alginate gel solution on the right side only.

Cystoscopic and radiographic examinations were performed at two, four,and six months after treatment. Cystoscopic examinations showed a smoothbladder wall. Cystograms showed no evidence of reflux on the treatedside and persistent reflux in the uncorrected control ureter in allanimals. All animals had a successful cure of reflux in the repairedureter without evidence of hydronephrosis on excretory urography.

At the time of sacrifice, gross examination of the bladder injectionsite showed the presence of bladder muscle cell-alginate composites inthe subureteral region. Microscopic analyses of the tissues surroundingthe injection site showed no inflammation. Tissue sections from thebladder, ureters, lymph nodes, kidneys, liver and spleen showed noevidence of alginate migration or granuloma formation.

SUMMARY OF EXPERIMENTAL DATA

Autologous bladder muscle cells can be readily harvested, expanded invitro, and injected cystoscopically. The cells survive and form a musclenidus which is non-antigenic. This system is able to correct refluxwithout any evidence of obstruction.

Modifications and variations of the present invention will be obvious tothose skilled in the art from the foregoing detailed description. Suchmodifications and variations are intended to come within the scope ofthe appended claims.

We claim:
 1. A method for treating conditions which require thereconstruction of an anatomical area selected from the group consistingof the thoracic region, gastrointestinal tract, urinary tract, andreproductive tract comprising injecting into a patient in need oftreatment thereof at a site in the anatomical area a suspension ofsmooth muscle cells in a biodegradable non-proteinaceous polymersolution that forms an ionically crosslinked hydrogel having the cellsdispersed therein when injected in vivo, which becomes a non-migratory,volume stable tissue mass.
 2. The method of claim 1 wherein the polymeris crosslinkable by temperature or pH and is selected from the groupconsisting of polysaccharides, polyphosphazines, polyacrylates, andpolyethylene oxide-polypropylene glycol block copolymers.
 3. The methodof claim 2 wherein the polymers have acidic side groups that can bereacted with cations and are selected from the group of polymersconsisting of poly(phosphazenes), poly(acrylic acids), poly(methacrylicacids), copolymers of acrylic acid and methacrylic acid, poly(vinylacetate), sulfonated polymers, and copolymers having acidic side groupsformed by reaction of acrylic or methacrylic acid and vinyl ethermonomers or polymers.
 4. The method of claim 2 wherein the polymers havebasic side groups that can be reacted with anions and are selected fromthe group of polymers consisting of poly(vinyl amines), poly(vinylpyridine), poly(vinyl imidazole), and imino substitutedpolyphosphazenes.
 5. The method of claim 2 wherein the polymer isselected from the group consisting of alginate and hyaluronic acid. 6.The method of claim 1 wherein the muscle cells are isolated from thepatient.
 7. The method of claim 1 wherein the condition isvesicoureteral reflux.
 8. The method of claim 1 wherein the condition isincontinence.
 9. The method of claim 1 wherein the area which requiresreconstruction is in the thoracic region.
 10. The method of claim 1wherein the area which requires reconstruction is in the uppergastrointestinal tract.
 11. A method for obstructing a lumen or tube ina patient in need of treatment thereof comprising injecting into thelumen or tube in the patient a suspension of autologous muscle cells ina biodegradable polymer solution that forms a hydrogel having the cellsdispersed therein which becomes a non-migratory, volume stable tissuemass.
 12. The method of claim 11 wherein the polymer can be crosslinkedby a change in pH, addition of multivalent ion, or a change intemperature.
 13. The method of claim 11 wherein the polymer is asynthetic polymer.
 14. The method of claim 13 wherein the polymers areselected from the group consisting of poly(phosphazenes) that can becrosslinked by reaction with cations, poly(acrylic acids),poly(methacrylic acids), copolymers of acrylic acid and methacrylicacid, poly(vinyl acetate), sulfonated polymers, copolymers having acidicside groups formed by reaction of acrylic or methacrylic acid and vinylether monomers or polymers, poly(vinyl amines), poly(vinyl pyridine),poly(vinyl imidazole), and imino substituted polyphosphazenes.
 15. Themethod of claim 11 wherein the polymer is selected from the groupconsisting of fibrin, collagen, alginate, and hyaluronic acid.
 16. Themethod of claim 11 wherein the tube is a fallopian tube.
 17. The methodof claim 1 wherein the area which requires reconstruction is a regionadjacent to or a part of the rectum.
 18. The method of claim 1 whereinthe area which requires reconstruction is a region adjacent to or a partof the esophogus.
 19. The method of claim 1 wherein the area whichrequires reconstruction is adjacent to or a part of the ureter.